Metastatic melanoma, a tumor of pigment-producing cells of the skin, is a highly aggressive disease with limited therapeutic options. Several lines o evidence indicate that melanoma cells revert to a neural crest stem cell fate, suggesting an avenue for therapeutic intervention. A previous screen of 2000 compounds for suppressors of the neural crest lineage in zebrafish embryos pointed to inhibitors of dihydroorotate dehydrogenase (DHODH). The DHODH inhibitor leflunomide was shown to suppress neural crest by inhibiting transcriptional elongation of genes required for neural crest development. Leflunomide also suppresses the growth of human melanoma cell lines and murine xenograft tumors, and it is currently being tested in clinical trials in combination with the BRAF inhibitor PLX4720. The goal of this project is to identify additional chemical suppressors of the neural crest and test them for antitumor activity in melanoma model systems. I propose to screen on a larger scale by developing a protocol for the culture of neural crest lineage from transgenic zebrafish embryos in which the neural crest is fluorescently labeled. In contrast to traditional whole embryo analysis, an in vitro system is readily adapted to entirely automated high-throughput screening. Once assay development is complete, I expect that 20,000 compounds can be screened in 12-14 weeks. Screening of zebrafish embryonic cells in culture has produced validated results in our lab. A fluorescent reporter-based screen for suppressors of the muscle lineage identified compounds that suppress the muscle lineage, 65% of which were validated in whole embryos. I will use cultured neural crest cells as a tool to identify potential suppressors of the neural crest lineage in vivo, which will be evaluated by in situ hybridization for neural crest markers. Validated compounds will be assayed for effects on apoptosis, proliferation, invasion, and neural crest identity of human melanoma cell lines and zebrafish primary tumor cultures. Compounds will be categorized based on activity in these assays and known or predicted biomolecular targets. One category of compounds will be selected for evaluation of antitumor activity in murine xenograft tumors and an mitf:BRAF(V600E); p53 -/- zebrafish model of melanoma developed in our lab. The proposed project has the potential to identify novel therapeutic targets for melanoma, a disease for which resistance to current therapies poses a major problem. Compounds with lineage specific effects have the potential treat melanoma with a high therapeutic index and broad applicability, and identification of pathways important for neural crest differentiation and maintenance will provide insight into both embryonic development and melanoma tumorigenesis.